The present invention relates to a gene for proline transporter in rice.
It is known that some plants, including halophytes, when they are under salt stress, accumulate proline in their bodies. It is considered that the proline accumulated serves as a compatible osmolyte, regulating the intracellular osmotic pressure and keeping water in the plant body. In plants, proline is synthesized from glutamic acid by two reactions catalyzed by two enzymes, namely xcex941-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and xcex941-pyrroline-5-carboxylate reductase (P5CR), respectively.
When such plants undergo water stress (state in which water absorption is difficult), such as salt stress or water deficit, the levels of P5CS activity and P5CS gene expression increase. In that case, the levels of P5CR activity and P5CR gene expression remain almost constant and are low. This indicates that P5CS is a rate-limiting factor in the proline synthesis under water stress (Yoshiba et al., Plant J. 7:751-760 (1995)).
Kishor et al. succeeded in increasing the salt tolerance of tobacco by introducing the P5CS gene of mothbean thereinto to thereby cause overexpression of that gene, indicating that gene manipulation involving the P5CS gene is effective in producing salt tolerant crop plants (Kishor et al., Plant Physiol. 108:1387-1394 (1995)).
Not only the proline synthesis but also proline transport is involved in the accumulation of proline under water stress. As regards the proline transport, two proline transporter genes have been cloned from Arabidopsis thaliana and three from tomato. The transporters, unlike other amino acid permeases, transport proline alone selectively.
Upon amino acid sequence comparison, the two Arabidopsis proline transporter genes are shown to be very close to each other. They differ, however, in the mode of gene expression from each other. One is the Arabidopsis proline transporter 1 (AtProT1) gene, which is expressed constantly at a low level throughout tissues, and the other is the Arabidopsis proline transporter 2 (AtProT2) gene, which is scarcely expressed under normal growth conditions but whose expression is swiftly and intensely induced under salt stress conditions. Therefore, the latter gene is considered to be a transporter gene prepared exclusively for water stress and serving to transport proline as a compatible osmolyte (Rentsch et al., The Plant Cell 8:1437-1446 (1996)).
Among the three proline transporter genes cloned from tomato, the proline transporter 1 (LeProT1) gene has been analyzed to a good extent with respect to its function and it is known that it is intensely expressed in the process of pollen maturation and LeProT1 transports proline to and cause accumulation thereof in pollen. It is a characteristic feature of LeProT1 that it transports not only proline but also glycine betaine and like compatible osmolytes induced by water stress. At present, no report is available of the functions of the proline transporter 2 (LeProT2) and proline transporter 3 (LeProT3). However, since the genes encoding them belong to the same gene family as that to which the LeProT1 belongs, they are considered to be involved in amino acid transport to pollen and/or in stress-responding transport, like AtProT2 (Schwacke et al., The Plant Cell 11:377-391 (1999)).
For proline accumulation under water stress, proline synthesis and transport of the proline synthesized are both important. For plants to cope with rapid environmental changes such as drought and/or salt stress, it is effective to prompt proline synthesis as far as possible and transport proline to tissues requiring it. If, for such purposes, the P5CS gene can be manipulated to realize overexpression thereof and proline synthesis activation and, further the proline transporter gene or genes can be manipulated to realize overexpression thereof for transportation of the proline synthesized to tissues requiring the same, it will become possible to breed rice and other useful crop plants showing synergistic action and further having high salt tolerance and drought resistance. It is estimated that the proportion of saline soils resulting from drought and semidrought will increase more and more in the future as a result of destruction of the natural environment. Thus, breeding such tolerant or resistant crop plants as mentioned above plays an important role in solving the global food problem.
As far as the proline transporter genes in plants are concerned, only the corresponding cDNAs have so far been isolated from Arabidopsis thaliana and tomato. None has been isolated from monocots. Under such circumstances, the present invention provides a proline transporter gene in rice, which is a monocot. The present invention, by which a monocot proline transporter gene, which is quite unknown in the art, has been isolated, can be said to have achieved a novel technological advancement.
The present invention, which has been made with attention paid to the importance of such a rice proline transporter, has succeeded in solving a novel technical problem of controlling such transporter by gene manipulation. Namely, the present inventors newly raised the above technical problem so far not recognized in the art and approached the problem from various angles. Thus, they made intensive investigations in an attempt to isolate a relevant gene from rice seedling tissues and, as a result, successfully isolated a cDNA coding for the full length of a rice proline transporter gene from a cDNA library prepared by reverse transcription of mRNA.